Highly efficient transient expression of functional recombinant antibodies in lettuce

Negrouk, Valentine; Eisner, Galina; Lee, Hyung‐il; Han, Kaiping; Taylor, Dean; Wong, Hing C.

doi:10.1016/j.plantsci.2005.03.031

Cited by 55 publications

(37 citation statements)

References 9 publications

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“…In this study, the antiviral ChIFN-α was first introduced into plants by a transient expression system that offers a rapid testing of the exogenous gene con- Non-transgenic lettuce plants struction and the recombinant protein expression within a week before generating stably transformed plants (Negrouk et al, 2005). The IFN expression was in good agreement with the experimental results of HuIFN gene in transgenic plants from others (Ohya et al, 2001;Sawahel, 2002;Li et al, 2007).…”

Section: Resultssupporting

confidence: 63%

“…The resulted vector comprises cauliflower mosaic virus 35S promoter, the selectable NPT gene and report GUS gene, ChIFN-α gene (IFNR), Brassica napus napin signal peptide (tp) (Crouch et al, 1983) and NOS terminator (Fig.1). Recombinant Agrobacterium was maintained in a modified liquid yeast extract and beef (YEB) (Negrouk et al, 2005) with 100 mg/L kanamycin sulfate (Amresco, USA) and 20 mg/L rifampicin (Sigma, USA). Tumefaciens was grown to log phase and condensed to an OD 600 (optical density at 600 nm) of about 2.1 for transformation of vacuum infiltration.…”

Section: Transformation Vector and Cultivation Of Agrobacterium Tumefmentioning

confidence: 99%

“…The specific transformation of lettuce was performed according to the described method by Negrouk et al(2005) except that 0.065 MPa of vacuum degree was used in transformation. After infiltration, lettuce samples were cultivated for 72 h (16-h light/8-h dark photoperiod) at 22 °C (Vaquero et al, 1999).…”

Section: Transformation Of Lettuce Plantsmentioning

confidence: 99%

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Transient expression of chicken alpha interferon gene in lettuce

Song

Zhao

et al. 2008

J. Zhejiang Univ. Sci. B

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Abstract:We investigated the possibility of producing chicken alpha interferon (ChIFN-α) in transgenic plants. The cDNA encoding ChIFN-α was introduced into lettuce (Lactuca sativa L.) plants by using an agro-infiltration transient expression system. The ChIFN-α gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus (VSV) replication on chicken embryonic fibroblast (CEF). The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.

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Section: Resultssupporting

confidence: 63%

Section: Transformation Vector and Cultivation Of Agrobacterium Tumefmentioning

confidence: 99%

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Transient expression of chicken alpha interferon gene in lettuce

Song

Zhao

et al. 2008

J. Zhejiang Univ. Sci. B

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“…In the past few years, transient expression systems proved to be particularly attractive for the rapid accumulation of high amounts of recombinant proteins and in particular viral-based vector systems have been recently devised for high yield expression of full-size antibodies (Giritch et al 2006;Sainsbury et al 2009). On the other hand, transient expression based on Agroinfiltration of 35S promoter expression cassettes was also used to produce fully functional mAbs in tobacco, Nicotiana benthamiana, lettuce and in tomato fruit (Hull et al 2005;Negrouk et al 2005;Orzaez et al 2006;Strasser et al 2008;Vaquero et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration

et al. 2010

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It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15-20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).

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“…Agrobacterium-mediated transient expression analysis has become an alternative to overcome such problems and has been successfully used in several species for functional analysis of genes and promoters, protein production and localisation (Kapila et al 1997;van der Hoorn et al 2000;Yang et al 2000;Wroblewsky et al 2005;Sawers et al 2006). The method is rapid as it allows the assay of the transgene within a few days after the infiltration of Agrobacteria (Negrouk et al 2005;Wroblewsky et al 2005). Vacuum infiltration of Agrobacteria into the interior of the plant tissue has been efficiently used for transient assay of gene expression, and also for obtaining stably transformed plants by circumventing conventional time-consuming protocols (Negrouk et al 2005;Canche-Moo et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

Mani

Manjula

2010

Plant Cell Tiss Organ Cult

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The present study describes the successful development of vacuum infiltration method in the oomycete-resistant wild Piper sp., Piper colubrinum, as a rapid transient method for expression of GUS (b-Glucuronidase) reporter gene and introduction of hairpin vector for endogenous gene silencing. The GUS reporter gene construct pCAMBIA 1305.2 was used as a positive control to test the efficiency of vacuum infiltration strategy. Agrobacteria (EHA 105) harbouring GUS binary vector were vacuum-infiltrated into young detached in vitro leaf explants, which showed detectable GUS gene activity within 4 days of infiltration. This paper also reports for the first time the application of transient gene silencing in P. colubrinum by the delivery of in vitro synthesized hairpin vector construct (pHELLSGATE) containing endogenous serine threonine protein kinase (STPK) gene homologue into in vitro shoots. Introduction of hairpin vectors for the STPK gene into in vitro plantlets by vacuum infiltration resulted in significant reduction in transcript accumulation of the endogenous gene. The results indicate that transient gene silencing could be used as a rapid, preliminary high-throughput tool for P. colubrinum functional genomics.

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Highly efficient transient expression of functional recombinant antibodies in lettuce

Cited by 55 publications

References 9 publications

Transient expression of chicken alpha interferon gene in lettuce

Transient expression of chicken alpha interferon gene in lettuce

Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration

Optimization of Agrobacterium-mediated transient gene expression and endogenous gene silencing in Piper colubrinum Link. by vacuum infiltration

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